Project Summary Duchenne muscular dystrophy (DMD), the most common genetic muscle disease, is lethal with no cure at this point. Muscle pathology of DMD and its mouse model mdx5cv features chronic inflammation with predominant macrophage (MP) infiltration. Preclinical studies by our lab and others demonstrate that ameliorating muscle inflammation improves muscular dystrophy phenotype. It also improves local tissue environment to promote muscle regeneration and gene and cell engraftment. Tissue macrophages are functionally heterogeneous with diverse origins. They can be pro-inflammatory, pro-fibrotic, or pro-regenerative depending on the tissue environment and origins. While tissue inflammatory MPs are derived from blood monocytes (MOs), tissue resident MPs can originate from blood MOs and/or embryo. Murine peripheral blood MOs consist of two subsets, Ly6Chi and Ly6Clo cells, with corresponding subsets in humans. Ly6Chi cells (CCR2+/CX3CR1low) are inflammatory MOs, which enter tissues in response to injury via CC chemokine receptor 2 (CCR2) and then differentiate into inflammatory MPs. Within injured tissues, Ly6Chi MPs can switch into Ly6Clo MPs. Ly6Chi MOs may also contribute to tissue resident MPs at the steady state. Ly6Clo MOs (CCR2-/CX3CR1hi) patrol the vascular endothelial surface and may enter normal tissue via chemokine receptor CX3CR1 to replenish resident MPs. Embryo-derived tissue resident MPs are also Ly6Clo, and they persist into adult tissues through proliferative self-renewal. Our preliminary data show that both Ly6Chi and Ly6Clo subsets of MPs accumulate in mdx5cv skeletal muscle, and that CCR2 is essential to the muscle recruitment of Ly6Chi inflammatory MOs. Knockout of CCR2 diminishes intramuscular Ly6Chi MPs at all stages, but it only reduces Ly6Clo MPs at early stages. The reduction of intramuscular MPs at the early stages is accompanied by decreased muscle damage, reduced muscle fibrosis, and improved muscle function, which supports a pathogenic role for the intramuscular Ly6Chi MPs. However, the beneficial effects are lost at the late stage in the mdx5cv/Ccr2-/- mice after the expansion of intramuscular Ly6Clo MPs. Targeting Ly6Chi MP alone does not provide sustained benefits. We thus generate our central hypothesis that Ly6Clo MPs also play a pathogenic role in the mdx5cv diaphragm dystrophy, Ly6Clo MPs from different origins may contribute differently, and targeting the monocytic origins is therapeutically useful. We will test our hypothesis by three Specific Aims. Aim 1 will study the origins of skeletal muscle resident MPs at the normal steady state. Aim 2 will define the origins of intramuscular MPs in the mdx5cv diaphragm. Aim 3 will determine the effector and regulatory functions of intramuscular MPs derived from different origins in the mdx5cv diaphragm, and test the therapeutic potential of targeting monocytic origins. This project will address the key questions related to the intramuscular MPs, and the knowledge gained will be critical to the future development of novel monocyte/macrophage-based therapies for DMD.